It has been reported that platelet aggregation is induced by tumor cells in hematogenous metastasis of cancer cells. Most of cancer cells that invade into blood vessels are destroyed by an attack of the immune system of the host or by physical impact. However, it has been considered that the occurrence of platelet aggregation leads to protection of cancer cells against such a destructive process and enables metastasis of cancer cells (FIG. 13). On the other hand, it has been believed that platelet aggregation accelerates the adhesion of cancer cells to vascular endothelial cells, and the release of growth factors results in local proliferation of cancer cells. Further, capillary obstruction by clumps of platelets due to cancer cells also contributes to the acceleration of hematogenous metastasis.
By repeating experimental pulmonary metastasis of the mouse colon cancer cell line colon26, a high-metastatic strain NL-17 cell and a low-metastatic strain NL-14 cell have been established (Non-Patent Document 1). Further, a monoclonal antibody 8F11 has been constructed which exhibits high reactivity to NL-17 cells and shows low reactivity to NL-14 cells. In in vitro experiments, NL-17 cells caused platelet aggregation in mice, but such activity of NL-17 cells was inhibited by 8F11 antibodies. Further, in in vivo experiments, experimental pulmonary metastasis of NL-17 cells was inhibited by administration of 8F11 antibodies. Based on these findings, it has been suggested that NL-17 cells express a platelet-aggregating factor, which is recognized by 8F11 antibodies, to cause the aggregation of mouse platelets, consequently resulting in pulmonary metastasis. This platelet-aggregating factor was named later as “podoplanin” (also known as Aggrus).
Then, mouse podoplanin protein was purified from NL-17 cells using an 8F11 antibody column and a WGA column (Non-Patent Document 2). The purified podoplanin inhibited mouse platelet aggregation in the absence of plasma components in a concentration-dependent manner, and this aggregation reaction was completely inhibited by 8F11 antibodies.
Inventors of the present invention have now succeeded in gene cloning of podoplanin (Non-Patent Document 3). Podoplanin is a type I transmembrane protein having a C-terminal transmembrane domain. Human podoplanin, although having a low homology with mouse podoplanin, causes mouse platelet aggregation, whereas mouse podoplanin brings about platelet aggregation in a human. Through an epitope analysis of a neutralizing antibody of mouse podoplanin, 8F11 antibody, and detailed mutagenesis analyses, it has become clear that threonines (Thr) in three tandem repeats of EDxxVTPG (PLAG domain) form the active site for podoplanin-induced platelet aggregation and is conserved across species (Non-Patent Document 4). While sugar chains account for about a half of the molecular weight of podoplanin, it was determined by using glycosylation-deficient CHO mutant cells (Lec1, Lec2, Lec8) that sialic acid of an O-linked sugar chain added to Thr of the PLAG domain is the active center for platelet aggregation (Non-Patent Document 5).
Further, the present inventors have constructed a rat monoclonal antibody, NZ-1 antibody with high specificity for the purpose of purification of human podoplanin (Non-Patent Document 6). It has been seen that NZ-1 antibody is useful in Western blotting and flow cytometry as well as immunohistochemical staining, and is also utilized as an antibody having high sensitivity and specificity in immunoprecipitation. Since detailed structure analysis of sugar chain (particularly, O-linked sugar chain) using a mass spectrometer (MS) requires several tens of μg of a purified protein, screening of a cell line that expresses high level of human podoplanin was also carried out at the same time. As a result, using NZ-1 antibodies, human podoplanin was purified in large quantities from the human glioma cell line LN319 with high expression of human podoplanin (Non-Patent Document 7).
According to the detailed sugar chain structure analysis of human podoplanin, it has been elucidated that the active site for platelet aggregation of human podoplanin is a disialyl-core 1 structure added to Thr52 of a PLAG domain (Non-Patent Document 7).
Further, the present inventors have discovered that a receptor of podoplanin on platelets is CLEC-2 (C-type lectin-like receptor-2) of a C-type lectin-like receptor (Non-Patent Document 8). When Fc chimeras of CLEC-2 or membranous CLEC-2-expressing cells were constructed, specific binding between podoplanin and CLEC-2 was achieved. Further, podoplanin-induced platelet aggregation was inhibited by Fc chimeras of CLEC-2.
In addition, in order to confirm that podoplanin reacts with CLEC-2 through its PLAG domain, a variety of glycopeptides having an O-linked sugar chain added only to Thr52 of the PLAG domain were synthesized in vitro. As a result, only the glycopeptide having a disialyl-core 1 structure added to the PLAG domain exhibited high reactivity with CLEC-2 (Non-Patent Document 9).
NZ-1 antibodies inhibited binding of podoplanin to CLEC-2, and also inhibited podoplanin-induced platelet aggregation in a concentration-dependent manner. Further, tail vein injection of NZ-1 antibodies and podoplanin-expressing cells also exhibited significant inhibition of pulmonary metastasis (Non-Patent Document 9).
Further, the present inventors have synthesized 6 podoplanin Fc chimeras and 21 peptides and have confirmed that a minimal epitope of NZ-1 is AMPGAE and that 10 amino acids of GVAMPGAEDD are necessary for strong binding of podoplanin to NZ-1.
Further, anti-podoplanin antibodies (D2-40, AB3, 18H5 and rabbit polyclonal antibodies), which recognize other epitopes, did not inhibit interaction between podoplanin and CLEC-2 (Non-Patent Document 10).
From the results as above, human podoplanin has been shown to cause platelet aggregation through binding thereof to CLEC-2 and also carry out an important role in hematogenous metastasis of cancer, and thus it has been suggested that human podoplanin could be a cancer drug target.
Meanwhile, antibody pharmaceuticals using antibodies directed against disease-related targets have recently been developed. An antibody has a structure that two heavy chains (H chains) are associated with two light chains (L chains) stabilized via a pair of disulfide bonds. The heavy chain consists of a heavy-chain variable region VH, heavy-chain constant regions CH1, CH2 and CH3, and a hinge region positioned between CH1 and CH2. The light chain consists of a light-chain variable region VL and a light-chain constant region CL. Among these, a variable region fragment (Fv) consisting of VH and VL is a region which is directly involved in antigen binding and generates the diversity of antibodies. Further, an antigen-binding region consisting of VL, CL, VH and CH1 is referred to as a Fab region, and a region consisting of a hinge region, CH2 and CH3 is referred to as an Fc region.
The action mechanism of antibody pharmaceuticals is based on two biological activities of antibodies. One of them is a target antigen-specific binding activity, which is an activity neutralizing the function of a target antigen molecule through binding thereto. Functional neutralization of the target antigen molecule is exhibited through the Fab region. As an antibody pharmaceutical taking advantage of its neutralizing activity against an antigen molecule, infliximab or bevacizumab is known.
The other is a biological activity of an antibody known as an effector activity. The effector activity is exerted as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or direct induction of apoptosis, through an Fc region of an antibody. Examples of antibody pharmaceuticals that produce efficacy utilizing their effector activity include rituximab or trastuzumab (Non-Patent Document 11).
A neutralizing activity and an effector activity are independent of each other, and it cannot be said that an antibody with one activity always has the other activity. Further, since ADCC activity or CDC activity is dependent on subclasses of an antibody, it cannot be said that the antibody, even when having ADCC activity, has CDC activity and it cannot be said that the antibody, even when having CDC activity, has ADCC activity.
As an activity of antibody pharmaceutical, particularly an effector activity is regarded as important. For example, a human Fcy receptor IIIa has two types of polymorphism, one having a high affinity to rituximab and the other having a low affinity to rituximab. Among them superior clinical effects has been obtained in a non-Hodgkin's lymphoma patient with high-affinity polymorphism. Further, also in breast cancer therapy with trastuzumab, higher therapeutic effects are observed in a patient from whom a significantly high activity has been obtained in in vitro ADCC activity test using peripheral blood as an effector cell. These results suggest that ADCC activity is important for the development of clinically effective antibody pharmaceuticals. Accordingly, particularly regarding an anti-cancer agent, there is a need for an antibody pharmaceutical which is applicable for clinical use and exhibits a potent effector activity (Non-Patent Document 11).
As a measure of enhancing ADCC activity of an antibody, there are a method of modifying an amino acid sequence of an Fc region of an antibody and a method of controlling a structure of a sugar chain bound to an Fc region. However, such methods do not always bring about enhancement of ADCC activity. Further, even when ADCC activity is enhanced, since there is a variety of embodiments in modification of an amino acid sequence or control of a sugar chain structure, it is not easy to find a method of enhancing ADCC activity for a given antibody.
To date, podoplanin has been reported to exhibit high expression in brain tumor, mesothelioma, testicular tumor, ovarian cancer, and a variety of squamous cancers (oral cancer, pharynx cancer, larynx cancer, esophageal cancer, lung cancer, skin cancer, and uterine cervical cancer) (Non-Patent Documents 12 to 15). In particular, podoplanin is expressed in relation to malignancy in astrocytoma among brain tumors. Therefore, if there is an anti-podoplanin antibody having an effector activity such as ADCC activity or CDC activity as well as having a binding activity, it is expected that an anti-cancer action can be obtained also in such cancer.
However, as described above, with regard to NZ-1 antibody, only a platelet aggregation-neutralizing activity by the inhibition of binding of podoplanin to CLEC-2 has been confirmed hitherto, and thus involvement with hematogenous metastasis of cancer has merely been confirmed also in vivo.
Meanwhile, in research and development of antibody pharmaceuticals, immunogenicity in a human body is also a matter of concern. Monoclonal antibodies constructed with rodents such as mice or rats exhibit immunogenicity in a human body and may be contributory to attenuated effects or allergic reactions resulting from appearance of neutralizing antibodies. In order to avoid these disadvantages, a technique is being developed which renders an initial monoclonal antibody constructed using rodents into a chimeric antibody, a humanized antibody, or a fully human antibody with low antigenicity with respect to a human.
However, with regard to the method for preparing a chimeric antibody or a humanized antibody, there is no standardized method which is universally applicable to any antibody. Even when a chimeric antibody is constructed based on the antibody obtained from a different species, the resulting chimeric antibody may lose both a binding activity and an effector activity. Further, where a monoclonal antibody constructed with rodents such as mice or rats is made to be a chimeric antibody or a humanized antibody, it cannot be guaranteed that an equivalent activity can be obtained and an antibody having low antigenicity can be obtained.
As described above, regarding NZ-1 antibody, only a monoclonal antibody constructed with rats has been reported hitherto, and merely an activity of inhibiting binding of podoplanin and CLEC-2 to result in neutralization has been known for the constructed antibody. Further, an amino acid sequence of NZ-1 antibody, an amino acid sequence of a CDR or a gene sequence encoding the same has not been elucidated and there is no example specifically demonstrating a design of chimeric antibodies or the like.